TGFBR2 is a novel substrate and indirect transcription target of deubiquitylase USP9X in granulosa cells

Abstract

The ubiquitin‐specific peptidase 9 X‐linked (USP9X) is one of the highly conserved members belonging to the ubiquitin‐specific proteases (USPs) family, which has been reported to control substrates‐mediated biological functions through deubiquitinating and stabilizing substrates. Here, we have found that TGFBR2, the type II receptor of the transforming growth factor beta (TGF‐β) signaling pathway, is a novel substrate and indirect transcription target of deubiquitylase USP9X in granulosa cells (GCs). Mechanically, USP9X positively influences the expression of TGFBR2 at different levels through two independent ways: (i) directly targets and deubiquitinates TGFBR2, which maintains the protein stability of TGFBR2 through avoiding degradation mediated by ubiquitin‐proteasome system; (ii) indirectly maintains TGFBR2 messenger RNA (mRNA) expression via SMAD4/miR‐143 axis. Specifically, SMAD4, another substrate of USP9X, acts as a transcription factor and suppresses miR‐143 which inhibits the mRNA level of TGFBR2 by directly binding to its 3′‐untranslated region. Functionally, the maintenance of TGFBR2 by USP9X activates the TGF‐β signaling pathway, which further represses GC apoptosis. Our study highlights a functional micro‐regulatory network composed of deubiquitinase (USP9X), small noncoding RNA (miR‐143) and the TGF‐β signaling pathway, which plays a crucial role in the regulation of GC apoptosis and female fertility.

A regulatory network controlling ovarian granulosa cell death

“…Overexpression constructs pcDNA3.1-TGFBR2, pcDNA3.1-NORHA, pcDNA3.1-NORFA, and pcDNA3.1-NFIX were previously generated by our group [ 15 , 21 , 22 ]. The CMV promoter was isolated from the pcDNA3.1 expression vector and inserted between the Kpn I and Sac I restriction sites of the pGL3-basic vector (Promega, Madison, WI) to construct the pGL3-CMV vector.…”

“…Extraction and reverse transcription of total RNA were carried out as described previously [ 15 ]. Transcription levels were determined using AceQ qPCR SYBR Green Master Mix (#Q111-02, Vazyme, Nanjing, China) in a QuantStudio™ 7 Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…The detailed steps for harvest, extraction, and denaturation of total protein in GCs and western blot are described in our previous study [ 15 ]. GAPDH was used as an internal control.…”

“…Once bound to TGF-β1, the intracellular domain of TGFBR2 exhibits constitutive serine/threonine kinase activity [ 13 ]. In addition to TGF-β1, several modifying factors, including casitas B-lineage lymphoma (Cbl) [ 14 ], ubiquitin-specific peptidase 9 X-linked (USP9X) [ 15 ], asparagine-linked glycosylation 10 (ALG10) [ 8 ], and programmed death-ligand 1 (PD-L1) [ 16 ], also directly activate or maintain levels of activated TGFBR2 protein through neddylation, ubiquitination, glycosylation modifications, and lysosomal degradation. TGFBR2 promoter binding, transcription factors including SMAD4 [ 17 ] and GABPA [ 9 ] activate TGFBR2 transcription, whereas MYOCD [ 18 ] inhibits TGFBR2 transcription.…”

“…In sow granulosa cells (GCs), we previously demonstrated that TGFBR2 is a vital anti-apoptotic factor that is downregulated by miRNAs that bind to its 3′-UTR [ 12 , 15 ]. TGFBR2 can also be induced by binding SMAD4 to its promoter [ 17 ], and its protein levels are maintained by the deubiquitinase activity of USP9X [ 15 ] in sow GCs. In this study, using the 5′-UTR as the target region, we investigated the regulatory mechanism of TGFBR2 expression in sow GCs.…”